Leukocyte integrins play a major role in innate and adaptive immune mechanisms by mediating leukocyte migration, activation and antimicrobial functions. However, signaling mechanisms initiated by leukocyte integrins are poorly understood. We have recently shown, using syk-/- bone marrow chimeric mice, that the Syk tyrosine kinase is essential for integrin-mediated adherent activation of neutrophils. Syk is a central component in immunoreceptor (B- and T-cell-receptor and Fc-receptor) signaling. In these processes, Syk is recruited to the receptor complex by binding of its tandem SH2 domains to phosphorylated immunoreceptor tyrosine-based activation motifs (ITAMs). In the proposed study we aim to investigate the mechanisms through which Syk becomes activated by integrins. Based on our preliminary experiments, we hypothesize that, similar to immunoreceptor signaling, integrin signaling is also mediated by the recruitment of Syk to the receptor complex through SH2-mediated binding to phosphorylated ITAM motifs. This hypothesis will be tested by analysis of integrin-mediated functional responses and signaling pathways in neutrophils lacking two ITAM-bearing adapters, DAP12 and the Fc-receptor gamma-chain (FcRgamma). We wilt also test the existence of a signaling complex containing DAP12/FcRgamma, and Syk by coimmunoprecipitation and confocal microscopy to assess the co-localization of the two components. Furthermore, we will re-introduce functionally inactive point mutants of both Syk and DAP12/FcRgamma into the relevant knockout backgrounds by retroviral transduction of macrophages and hematopoietic stem cells. We will use inactivating mutations in the Syk SH2 domains as well as mutations removing critical tyrosine residues in the ITAM motifs of DAP12 and FcRgamma to test whether these mutants will reconstitute integrin signaling in neutrophils. If our hypothesis that integrin signaling follows the ITAM paradigm proves to be true, it would provide a novel insight into the mechanism of integrin signaling in phagocytes and it would improve our understanding of disease processes characterized by disregulation of the innate immune system. This research will be done primarily in Hungary at the Semmelweis University in Budapest, in collaboration with Attila Mocsai as an extension of NIH Grant # RO1 DK58066.